Dynamic Enolate Recognition in Aqueous Solution by Zinc(II) in a Phenacyl-Pendant Cyclen Complex:  Implications for the Role of Zinc(II) in Class II Aldolases

A zinc(II) complex of 1-(4-bromophenacyl)-1,4,7,10-tetraazacyclododecane, ZnL, has been synthesized to mimic the active center of class II aldolases. Its X-ray crystal structure showed that zinc(II) remains atop the four nitrogen atoms of cyclen (average Zn−N distance 2.170 Å) to bind with the carbonyl oxygen (Zn−O distance of 2.159(3) Å) and one H₂O (Zn−O distance of 2.130(3) Å). Crystal data:  monoclinic, space group P2₁/n (No. 14), with a = 12.221(8) Å, b = 11.052(7) Å, c = 18.194(9) Å, β = 97.12(3)°, V = 2438(5) ų, Z = 4, R = 0.041, and R_w = 0.046. The potentiometric pH titration of ZnL in aqueous solution disclosed dissociation of one proton with pK_a = 8.41 at 25 °C and I = 0.1 (NaClO₄), which yields a mixture of a hydroxide-bound complex (ZnL−OH^-) and an enolate-bound complex (ZnH_-1L) in a 3:1 ratio, as determined by UV spectrophotometric and ¹³C and ¹H NMR titrations. This is the first quantitative assessment of enolate formation promoted by a proximate zinc(II) ion near neutral pH in aqueous solution. The enolate-bound complex ZnH_-1L independently isolated by treatment of ZnL with an equivalent amount NaOMe in acetonitrile was fully characterized to compare with ZnL. The equilibrium between the ZnL−OH^- and ZnH_-1L varied with temperature (15, 25, and 35 °C), from which thermodynamic parameters of ΔG = 3.0 kJ mol^-1, ΔH = 8.7 kJ mol^-1, and ΔS = 19 J mol^-1 K^-1 at 25 °C were determined. Because of the facile enolization by zinc(II), the methylene hydrogen atoms (adjacent to the carbonyl) of ZnL were readily exchanged by deuterium under physiological conditions. The half-life at 25 °C for this H−D exchange at pD 7 (20 mM MOPS buffer) was determined to be 25 min by ¹H NMR measurements. Implications of the present results for the role of zinc(II) in the active center of class II aldolases are discussed.